Topoisomerase I Alone Is Sufficient to Produce Short DNA Deletions and Can Also Reverse Nicks at Ribonucleotide Sites
Year: 2015
Type: article
Abstract: Ribonucleotide monophosphates (rNMPs) are among the most frequent form of DNA aberration, as high ratios of ribonucleotide triphosphate:deoxyribonucleotide triphosphate pools result in approximately two misincorporated rNMPs/kb of DNA. The main pathway for the removal of rNMPs is by RNase H2. However, in a RNase H2 knock-out yeast strain, a topoisomerase I (Top1)-dependent mutator effect develops with accumulation of short deletions within tandem repeats. Proposed models for these deletions implicated processing of Top1-generated nicks at rNMP sites and/or sequential Top1 binding, but experimental support has been lacking thus far. Here, we investigated the biochemical mechanism of the Top1-induced short deletions at the rNMP sites by generating nicked DNA substrates bearing 2′,3′-cyclic phosphates at the nick sites, mimicking the Top1-induced nicks. We demonstrate that a second Top1 cleavage complex adjacent to the nick and subsequent faulty Top1 religation led to the short deletions. Moreover, when acting on the nicked DNA substrates containing 2′,3′-cyclic phosphates, Top1 generated not only the short deletion, but also a full-length religated DNA product. A catalytically inactive Top1 mutant (Top1-Y723F) also induced the full-length products, indicating that Top1 binding independent of its enzymatic activity promotes the sealing of DNA backbones via nucleophilic attacks by the 5′-hydroxyl on the 2′,3′-cyclic phosphate. The resealed DNA would allow renewed attempt for repair by the error-free RNase H2-dependent pathway in vivo . Our results provide direct evidence for the generation of short deletions by sequential Top1 cleavage events and for the promotion of nick religation at rNMP sites by Top1. Background: DNA deletions at short repeat sites containing ribonucleotides are Top1-dependent. Results: Top1 generates DNA deletions at short repeat sites in vitro and also reverses nicks at ribonucleotide sites. Conclusion: The impact of Top1 on the ribonucleotide sites is both negative and positive. Significance: We elucidate the detailed mechanism of Top1 interacting with ribonucleotide sites, the most frequently misincorporated non-canonical nucleotides.
Source: Journal of Biological Chemistry
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
OpenAlex ID: W2012107742